DNA analysis method and DNA analyzer

ABSTRACT

Disclosed is a DNA analysis method and a DNA analyzer whose signal intensity is not lowered even when a material at a higher density is measured. There is supplied dATP, dTTP, dGTP, or dCTP from a dATP solution vessel, a dTTP solution vessel, a dGTP solution vessel, or a dCTP solution vessel, and this causes an extension reaction of a double-stranded DNA immobilized to a bead, to yield pyrophosphoric acid. The pyrophosphoric acid is converted into a redox compound by the actions of a reagent and an enzyme contained in a reaction buffer in a reaction buffer vessel. The redox compound causes a variation in surface potential of a measuring electrode bearing an electrochemically active material immobilized thereto through an insulating molecule, and this variation causes a variation in drain current of a field-effect transistor electrically connected to the measuring electrode. Thus, the extension reaction is detected.

CLAIM OF PRIORITY

The present application is a Divisional of U.S. application Ser. No.12/068,206 filed Feb. 4, 2008. Priority is claimed from the U.S.application Ser. No. 12/068,206 filed Feb. 4, 2008, which claimspriority from Japanese application JP 2007-077294 filed on Mar. 23,2007, the content of which is hereby incorporated by reference into thisapplication.

FIELD OF THE INVENTION

The present invention relates to a method and a system that candetermine base sequences of DNAs in trace amounts with high sensitivity.

BACKGROUND OF THE INVENTION

With advancing base sequence analysis technologies such as DNAsequencers, complete genome sequences have been analyzed in a variety ofbiological species such as the human. These analyzed genome sequencesbelong to specific individuals, and studies on differences in genomesequence among individuals have been launched as the next stage.However, it is difficult to analyze complete genome sequences ofrespective individuals, because it takes enormous time and cost for thecurrent base sequence analysis technologies to analyze complete genomesequences in an individual-to-individual manner. Demands have thereforebeen made to provide a base sequence analysis technology that cananalyze genome sequences in a short time at low cost. Specifically, DNAsequencers desirably have higher throughputs and analyze samples insmaller amounts.

For enabling DNA sequencers to have higher throughputs, M. Margulies etal. discloses a process using bead handling and pyrosequencingtechnologies in combination in Nature 437, 376-380 (2005). According tothis process, DNA to be analyzed is immobilized to beads, and 45×10⁴beads are integrated (packed) and concurrently subjected topyrosequencing. Thus, a base sequencing rate per one base can beincreased. The pyrosequencing is a base sequence analysis technologyutilizing that pyrophosphoric acid is released when a deoxynucleotidetriphosphate (dNTP) is taken into a double-stranded DNA as a result of asynthesis reaction (extension reaction) of the double-stranded DNAcatalyzed by a DNA polymerase. A DNA (sample DNA) whose base sequence isto be analyzed is hybridized with a DNA (primer DNA) deciding the startpoint (origin) of complementary strand synthesis. Four dNTPs (dATPαS,dTTP, dGTP, and dCTP) are sequentially fed to the hybridized DNA in thepresence of a DNA polymerase, and, when an extension reaction occurs,pyrophosphoric acid is formed in a number corresponding to the number ofextended bases. The formed pyrophosphoric acid is converted to lightemission by an enzymatic reaction by the catalysis of luciferase, andthe light emission is detected with a photoelectric transducer. The basesequence of the sample DNA can be determined by detecting the presenceor absence of an extension reaction and/or determining the quantity oflight emission when different dNTPs are added. In this process, dATPαSis generally used as a dNTP instead of dATP, because dATP and ATP areresemble with each other in structure, and background light is nottrivial when dATP is used as the dNTP.

On the other hand, electrical DNA sequencings and single nucleotidepolymorphism (SNP) typings have been conducted using, for example,field-effect transistor (FET) sensors and pH sensors instead of usingoptical detection techniques. A DNA sequencer using FET sensors detectsan extension reaction by the catalysis of a DNA polymerase as avariation in surface potential (T. Sakata et. al., Angew. Chem. Int. Ed.45, 2225-2228 (2006)). Specifically, a sample DNA is hybridized with aprimer DNA that has been immobilized to a surface of a FET sensor, andfour dNTPs (dATPαS, dTTP, dGTP, and dCTP) are sequentially fed theretoin the presence of a DNA polymerase. When an extension reaction occurs,a surface potential of the FET sensor decreases, because phosphatesgroups in side chain of the DNA have negative charges in an aqueoussolution, and when the number of bases of the DNA immobilized to thesurface of the FET sensor increases, the amount of negative charges onthe surface of the FET sensor increases. By detecting the variation insurface potential using the FET sensor, the base sequence of the sampleDNA can be determined.

In a pyrophosphate-detecting sensor using a pH sensor (Japanese PatentNo. 3761569), pyrophosphoric acid formed as a result of a nucleic-acidamplification reaction, such as a polymerase chain reaction (PCR), isenzymatically converted into a hydrogen ion, and the increased hydrogenion is detected with the pH sensor. Whether a polymerase chain reaction(PCR) is detected from how much the pH varies, and a single nucleotidepolymorphism (SNP) of a sample DNA is determined.

SUMMARY OF THE INVENTION

One of embodiments of the present invention provides a deoxyribonucleicacid (DNA) analysis method comprising the steps of: providing a samplecontaining a DNA to be analyzed and a primer hybridized to the DNA;carrying out an extension reaction of the primer in the presence ofdeoxyribonucleotide triphosphates (dNTPs) and a DNA polymerase so that aprimer extension reaction product is formed; converting pyrophosphoricacid formed as a result of the extension reaction into a redox compound;and electrically detecting the redox compound.

And one of embodiments of the present invention also provides a DNAanalysis system comprising: a vessel bearing an immobilized nucleic acidor containing an article bearing an immobilized nucleic acid, whereinthe vessel is configured to receive a liquid; one or more measuringelectrodes that are in contact with the liquid to be contained in thevessel; an electrochemically active material that is immobilized to asurface of the measuring electrode through an insulating molecule; areference electrode that is in contact with the liquid; one or morepotentiometers that measure interface potentials of the one or moremeasuring electrodes; a first feeder that feeds dATP or an analoguethereof to the vessel; a second feeder that feeds dGTP or an analoguethereof to the vessel; a third feeder that feeds dCTP or an analoguethereof to the vessel; and a fourth feeder that feeds dTTP or ananalogue thereof to the vessel.

Pyrosequencers can have higher throughputs by reducing diameters ofbeads to which primer DNAs are immobilized and using the beads at ahigher density. However, when a smaller amount of a sample DNA is usedalong with an increasing density of the beads, the quantity of lightemission decreases, because the pyrosequencing is based on thatpyrophosphoric acid formed as a result of a DNA extension reaction isconverted to light emission, and the light emission is detected.Specifically, when beads have smaller diameters and used at a higherdensity, the detection sensitivity is lowered, an expensive detector isrequired, and the entire system is increased in size and cost. Inaddition, dATPαS used in pyrosequencers is expensive as much as 100times the cost of dATP, and this causes increased cost for the basesequence analysis.

DNA sequencers using FETs fundamentally carry out sequencing bymeasuring potentials and can advantageously maintain their sensitivitieseven when sample volume decreases. In addition, their FET sensors can beeasily integrated at a higher density, because they do not requirecomplicated detection systems. However, a variation in surface potentialper one base extension reaction decreases with a proceeding extensionreaction of a double-stranded DNA and with an increasing distancebetween an extension reaction site and the surface of the sensor. Thisis because the effect of a negative charge on the surface potentialvaries depending on the distance between the surface of the FET sensorand the charge. Specifically, a detectable base length variessignificantly depending on the Debye length, and a detectable baselength is about ten bases even in a specially low-concentration buffer(2.5 mM) according to the known technology. The Debye length in thiscase is about 1 nm, and a theoretical detection limit is about thirtybases in consideration of the size of one base (0.34 nm). Accordingly,it is difficult to use the technology in general sequencing.

In contrast, when pyrosequencing is conducted using aphosphate-detecting sensor including a pH sensor, the sensitivity is notlowered even when sample volume decreases. This is because not theamount but the concentration of pyrophosphoric acid is detected fordetecting pyrophosphoric acid formed as a result of a DNA extensionreaction. In addition, this sensor is not limited in detectable baselength in contrast to the DNA sequencer using FETs and is expected toexhibit a detectable readout base length equivalent to that inpyrosequencing in which light emission is detected. It is difficult,however, to constitute a DNA sequencer using this sensor, because pH mayvary by the action of a reagent used in DNA sequencing and pH variationcaused by the formed pyrophosphoric acid may be buffered by the actionof a buffer.

Under these circumstances, according to an embodiment of the presentinvention, pyrophosphoric acid formed as a result of a DNA extensionreaction is enzymatically converted into a redox compound and the redoxcompound is electrically detected. An electrochemically active materialis immobilized to a surface of an electrode (measuring electrode)through an insulating molecule to reduce variation in potential due to areaction or adsorption of a foreign substance on the surface of theelectrode. The electrochemically active material to be immobilized isoptimized according to the redox compound to be detected. According toanother embodiment of the present invention, an insulating-gatefield-effect transistor is used as a potentiometer, which field-effecttransistor is arranged on the same substrate as the measuring electrode.To integrate detectors at a higher density so as to carry out DNAsequencing at a high throughput, an integrated circuit is prepared byarranging measuring electrodes and insulating-gate field-effecttransistors on the same substrate, and data are converted into serialdata using the integrated circuit.

According to an embodiment of the present invention, pyrophosphoric acidis converted into a redox compound and a variation in concentration ofthe redox compound is detected as a variation in surface potential.Thus, sensors can be used at a higher density, and smaller amounts ofsamples can be analyzed without reducing the detection sensitivity.Measurements using dATP, more inexpensive than dATPαS, can be conducted,because ATP is not directly involved in the reaction system, andbackground signals due to dATP do not occur. The signal intensity hereindoes not decrease even with a proceeding extension reaction of adouble-stranded DNA, because the signal intensity does not varydepending on the extension reaction site of the double-stranded DNA. Inaddition, this process is resistant to effects of buffers and reagents,because pyrophosphoric acid as a product of a DNA extension reaction isconverted into a redox compound. A leakage current causing the reductionin sensitivity can be reduced, and, concurrently, a variation inpotential due to a reaction and adsorption of a foreign substance on thesurface of the electrode can be reduced by immobilizing anelectrochemically active material to the surface of the electrodethrough an insulating molecule. The sensitivity can be increased byselecting an electrochemically active material to be immobilizedaccording to a redox compound to be detected. A down-sized system can beconstructed at low cost by using, as a potentiometer, an insulating-gatefield-effect transistor arranged on the same substrate as a measuringelectrode. For a higher throughput of the system, the number ofinterconnections can be reduced and the system can have a simplifiedconfiguration by converting data from the insulating-gate field-effecttransistor arranged on the same substrate as the measuring electrodeinto serial data in an integrated circuit arranged on the samesubstrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram illustrating a DNA analysis system accordingto an embodiment of the present invention;

FIG. 2 is a flow chart illustrating a DNA analysis method using a DNAanalysis system according to an embodiment of the present invention;

FIG. 3 is a block diagram illustrating an example of a circuit diagramof a FET sensor for use in a DNA analysis system according to anembodiment of the present invention;

FIGS. 4A and 4B are explanatory diagrams of a system for evaluating theimmobilization of an electrochemically active material to a surface of ameasuring electrode through an insulating molecule;

FIG. 5 is a diagram showing an effect of the immobilization of anelectrochemically active material to a surface of a measuring electrodethrough an insulating molecule;

FIG. 6 is a diagram showing an effect of the immobilization of anelectrochemically active material to a surface of a measuring electrodethrough an insulating molecule;

FIG. 7 is an explanatory diagram of a system for evaluating theimmobilization of an electrochemically active material to a surface of ameasuring electrode through an insulating molecule;

FIG. 8 is a diagram showing an effect of the immobilization of anelectrochemically active material to a surface of a measuring electrodethrough an insulating molecule;

FIG. 9 is a diagram showing an effect of the immobilization of anelectrochemically active material to a surface of a measuring electrodethrough an insulating molecule;

FIG. 10 is a diagram showing an effect of the immobilization of anelectrochemically active material to a surface of a measuring electrodethrough an insulating molecule.

FIG. 11 is a graph showing data of an analysis using a DNA analysissystem according to an embodiment of the present invention by way ofexample;

FIG. 12 is a graph showing how a single nucleotide polymorphism isdetected; and

FIG. 13 is a graph showing how an apparent nucleic acid concentrationvaries depending on the radius of a bead bearing an immobilized nucleicacid.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Some embodiments of the present invention will be illustrated below withreference to the attached drawings.

FIG. 1 is a block diagram illustrating a nucleic acid sequence analysissystem according to an embodiment of the present invention. The analysissystem according to this embodiment includes a measuring unit 101, asignal processing circuit 102, and a data processor 103. The measuringunit 101 includes a washing buffer vessel 104, a reaction buffer vessel105, a dATP solution vessel 106, a dTTP solution vessel 107, a dGTPsolution vessel 108, a dCTP solution vessel 109, a washing buffer supplyvalve 110, a reaction buffer supply valve 111, a dATP solution supplyvalve 112, a dTTP solution supply valve 113, a dGTP solution supplyvalve 114, a dCTP solution supply valve 115, a measuring cell 116,separators 117, beads 118, measuring electrodes 119, field-effecttransistors 120, a reference electrode 121, and a discharge liquidvessel 122. The supplies of the respective solutions can be controlledby opening or closing the respective valves. The measuring cell 116 ispartitioned to two or more (n) spaces by the separators 117. Each spaceincludes one set of a bead 118, a measuring electrode 119, and afield-effect transistor 120. A probe DNA is immobilized to a surface ofeach bead 118, and a target DNA hybridizes with the probe DNA. In casethat a sample is RNA, complimentary DNA (cDNA) of the RNA can be used.The beads may be, for example, generally-used polystyrene beads ormagnetic beads. The surfaces of the beads are modified typically withcarboxyl group, amino group, maleimido group, hydroxyl group, biotin, oravidin. Probe DNAs modified typically with a corresponding moiety suchas amino group, carboxyl group, SH group, silanol group, avidin, orbiotin are immobilized thereto. Fine gold particles, for example, may beused instead of beads. In this case, probe DNAs modified with afunctional group can be immobilized by coating the fine gold particleswith a molecule having a corresponding functional group. Anelectrochemically active material is immobilized to a surface of themeasuring electrode 119 through (by the interposition of) an insulatingmolecule. The measuring electrode 119 may be a noble metal electrode,such as a gold electrode, or a carbon electrode. The reference electrode121 is in contact with a solution in the discharge liquid vessel 122.

A washing buffer in the washing buffer vessel 104 herein was reducednicotinamide adenine dinucleotide (NADH). A reaction buffer in thereaction buffer vessel 105 herein was a solution of DNA polymerase,pyruvate orthophosphate dikinase (PPDK), lactate dehydrogenase,phosphoenolpyruvic acid (PEP), adenosine monophosphate (AMP), and NADHin Tris-HCl buffer. Solutions in the dATP solution vessel 106, dTTPsolution vessel 107, dGTP solution vessel 108, and dCTP solution vessel109 were solutions of dATP, dTTP, dGTP, and dCTP, respectively, inTris-HCl buffer. The reference electrode 121 herein was a Ag/AgClreference electrode containing a saturated potassium chloride solutionas an internal fluid. However, any reference electrode will do, as longas it has a variation in potential sufficiently smaller than thevariation in potential corresponding to one base extension.

In the present embodiment, the reference electrode 121 was in contactwith a solution in the discharge liquid vessel 122. However, thereference electrode 121 can be arranged at any position in the system aslong as it is in contact with a solution in a measuring cell. The beadsherein were polystyrene beads having a diameter of 50 μm and containingterminal carboxyl groups. A probe DNA was immobilized to the beads bymixing the beads with an amino-modified probe DNA and addingN-hydroxysuccinimide (NHS) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) thereto to therebychemically bind the probe DNA with the beads. A gold electrode was usedas the measuring electrode 119. The insulating molecule and theelectrochemically active material used herein were11-amino-1-undecanethiol (11-AUT) and pyrroloquinoline quinone (PQQ),respectively. A self-assembled monolayer of 11-AUT was formed on thegold electrode using a solution of 11-AUT. A mixture of PQQ, NHS, andEDC was added dropwise onto this electrode, a reaction was conductedovernight to immobilize PQQ to the electrode through a chemical bondbetween an amino group of 11-AUT and a carboxyl group of PQQ.

Procedural steps are illustrated in FIG. 2 by way of example. Initially,the washing buffer supply valve 110 was opened (202), the measuring cell116 was filled with a washing buffer (potential initializer) (203), andthen the washing buffer supply valve 110 was closed (204). By thisprocedure, the electrochemically active material immobilized to thesurface of the measuring electrode 119 was reduced. Next, the reactionbuffer supply valve 111 was opened (205), the measuring cell was filledwith the reaction buffer (206), and the reaction buffer supply valve 111was then closed (207). A constant voltage V_(G) was applied to thereference electrode 121, and drain currents of respective field-effecttransistors were measured (208). The respective measured drain currentswere indicated as I_(D) (1, n) (“n” is a number assigned to afield-effect transistor). The reaction buffer supply valve 111 and thedNTP solution supply valve 112, 113, 114, or 115 were opened (209), themeasuring cell 116 was filled with a mixture of the reaction buffer andthe dNTP solution (210), and the reaction buffer supply valve 111 andthe dNTP solution supply valve 112, 113, 114, or 115 were closed (211).As the dNTP were sequentially used dATP, dTTP, dCTP, and dGTP (201, 213to 217). A constant voltage V_(G) was applied to the reference electrode121, and drain currents of the respective field-effect transistors weremeasured (212). The measured drain currents herein were indicated asI_(D)(2, n) (“n” is a number assigned to a field-effect transistor), inwhich ΔI_(D)(n)=I_(D)(2, n)−I_(D)(1, n), in which ΔI_(D) represents avariation in drain current of a field-effect transistor as a result ofsupply of a dNTP. The procedure was returned to the operation of openingthe washing buffer supply valve 110 (202), and measurements wererepeatedly conducted sequentially using dATP, dTTP, dCTP, and dGTP as adNTP.

Alternatively, dATP, dTTP, dGTP, and dCTP may be dissolved in a reactionbuffer in the reaction buffer vessel 105 for use as a dATP solution inthe dATP solution vessel 106, a dTTP solution in the dTTP solutionvessel 107, a dGTP solution in the dGTP solution vessel 108, and a dCTPsolution in the dCTP solution vessel 106, respectively. In this case,the operation of opening the dNTP solution supply valve 112, 113, 114,or 115 is employed instead of the operation of opening the reactionbuffer supply valve 111 and the dNTP solution supply valve 112, 113,114, or 115 (209). Any order of dATP, dTTP, dCTP, and dGTP used as thedNTP will do, as long as each dNTP is used every four times. Instead ofa dNTP, any substance will do, as long as it is taken into a synthesisreaction of a double-stranded DNA by the catalysis of a DNA synthetasein a manner specific to a base sequence. An example of the substance isan analogue except with sulfur atom substituting a part of molecule(dNTPαS, in which the 4′-position of pentose is substituted with sulfur(N. Inoue et. al., Nucleic Acids Research, 3476-3483, 34, 2006)). Thewashing buffer is used for initializing the surface potential of anelectrode which has been varied by the action of a redox compoundconverted from pyrophosphoric acid. In this embodiment, the surfacepotential is increased due to an extension reaction, but it is reducedby the action of a reducing material in the washing buffer. Thus,another measurement of an extension reaction can be conducted. Asolution containing a reducing material such as a thiol compound insteadof the above-mentioned material may be used. The surface potential maybe reduced in some combinations of a redox compound and an enzyme in thereaction buffer. In this case, the washing buffer may be a solutioncontaining an oxidant such as hydrogen peroxide or potassiumferrocyanide.

FIG. 3 is a circuit diagram illustrating a field-effect transistor foruse in a nucleic acid sequence analysis system according to anembodiment of the present invention. The circuits shown in the circuitdiagram are preferably arranged or integrated on one substrate.Field-effect transistors are arrayed in a matrix, and a field-effecttransistor whose source/drain current-potential characteristics are tobe read out can be selected using an X-selector 301 and a Y-selector302. For example, when source/drain current-potential characteristics ofa field-effect transistor which is third from the left and second fromthe top are to be read out, interconnections the second from the top andthe third from the left are connected using the X-selector and theY-selector, respectively. Thus, the number of interconnections formeasuring potentials of a multiplicity of electrodes can be reduced.

A process of chemical reactions for detecting an extension reaction as avariation in potential in sequence analysis using a nucleic acidsequence analysis system according to an embodiment of the presentinvention will be illustrated below by way of example. A double-strandedDNA synthesis reaction on a bead yields pyrophosphoric acid. Thisreaction is catalyzed by a DNA polymerase.

Double-stranded DNA (“n” base length)+dNTP→Double-stranded DNA (“n+1”base length)+Pyrophosphoric acid

The formed pyrophosphoric acid is converted into pyruvic acid. Thisreaction is catalyzed by PPDK.

Pyrophosphoric acid+PEP+AMP→Pyruvic acid+ATP+Phosphoric acid

The formed pyruvic acid is converted into NAD. This reaction iscatalyzed by lactate dehydrogenase.

Pyruvic acid+NADH→Lactic acid+NAD

The formed NAD oxidizes PQQ immobilized to the surface of the electrode.

PQQ (reduced)+NAD

PQQ (oxidized)+NADH

This reaction is an equilibrium reaction, and the ratio of oxidized PQQto reduced PQQ is in accordance with the ratio of NAD to NADH.

Effects of the immobilization of an electrochemically active material toa surface of a measuring electrode through an insulating molecule willbe illustrated with reference to another embodiment. FIG. 4A illustratesan evaluation system for an untreated measuring electrode; and FIG. 4Billustrates an evaluation system for a measuring electrode bearing anelectrochemically active material immobilized thereto through aninsulating molecule. The evaluation system for an untreated measuringelectrode includes a potentiometer 401, a reference electrode 402, and ameasuring cell 403. The measuring cell 403 contains a measuring solution404, a gold electrode 405, and the reference electrode 402. Theevaluation system for a measuring electrode bearing an electrochemicallyactive material immobilized thereto through an insulating moleculeincludes a potentiometer 411, a reference electrode 412, and a measuringcell 413. The measuring cell 413 contains a measuring solution 416, agold electrode 417, and the reference electrode 412. The gold electrode417 bears an electrochemically active material 414 immobilized theretothrough an insulating molecule 415.

FIG. 5 shows how the potential difference between a reference electrodeand a gold electrode varies depending on the logarithm of the ratio inconcentration between redox compounds in the measuring solution (thelogarithm of the ratio of the potassium ferricyanide concentration tothe potassium ferrocyanide concentration). These data were measuredusing the evaluation systems in FIGS. 4A and 4B. In FIG. 5, a plotindicated by open triangle shows data measured by the evaluation systemin FIG. 4A; and a plot indicated by open circles shows data measured bythe evaluation system in FIG. 4B. The reference electrodes 402 and 412used herein were each a Ag/AgCl reference electrode containing asaturated aqueous potassium chloride solution as an internal fluid. Theelectrochemically active material 414 immobilized through the insulatingmolecule 415 was 11-ferrocenyl-1-undecanethiol (11-FUT). The measuringsolutions 404 and 416 were each a 0.1 M aqueous sodium sulfate solutioncontaining potassium ferricyanide and potassium ferrocyanide in a totalconcentration of 10 μM. The measurements were conducted at 25° C. Atlogarithms of the ratio in concentration between redox compounds withina range of −1 to 3, both the plot indicated by open triangles and theplot indicated by open circles well fit the straight line of 58 mV,showing that a potential occurs according to the Nernst's equation. Theplot indicated by open triangles, however, deviates from the straightline at logarithms of the ratio in concentration between redox compoundsof −2 or less and 4 or more, the range of potentials fitting thestraight line is 134 to 409 mV, and this corresponds to a range ofconcentrations of 4.7 digits. In contrast, the plot indicated by opencircles shows a range of potentials fitting the straight line of 80 to442 mV, and this corresponds to a range of concentrations of 6.2 digits.In other words, the dynamic range is increased by 1.5 digits byimmobilizing an electrochemically active material to a measuringelectrode through an insulating molecule.

When a measuring electrode does not have an insulating moleculeimmobilized thereto, the measuring electrode is not sufficientlyinsulated from a measuring solution. Accordingly, a leakage current onthe surface of the measuring electrode inhibits the measuring electrodefrom detecting a trace variation in concentration of a target materialto be measured. In contrast, by immobilizing an electrochemically activematerial to a measuring electrode through an insulating molecule, themeasuring electrode is highly insulated from a measuring solution, and aleakage current on the surface of the measuring electrode is reduced.Thus, the measuring electrode can detect a trace variation of the targetmaterial to be measured. In this case, molecules of theelectrochemically active material are preferably immobilized to themeasuring electrode through molecules of the insulating molecule havingidentical lengths. Thus, the insulating property between the measuringelectrode and the measuring solution can be maintained at satisfactorylevel, and variations in conditions of molecules of theelectrochemically active material uniformly affect the surface potentialof the measuring electrode.

Effects of the immobilization of an electrochemically active material toa surface of a measuring electrode through an insulating molecule willbe illustrated with reference to yet another embodiment of the presentinvention. FIG. 6 shows voltammograms measured using systems containingan untreated gold electrode and a gold electrode bearing an immobilizedinsulating molecule, respectively. The potentiostat used herein was theElectrochemical Analyzer ALS Model 611B. The reference electrode was aAg/AgCl reference electrode containing a saturated aqueous potassiumchloride solution as an internal fluid. The counter electrode was aplatinum wire. The measuring solution was a 0.1 M aqueous sodium sulfatesolution. In the voltammograms, a current varying in a scanningdirection represents an electrostatic capacity, and a slope versus anapplied potential represents a resistance of the electrode surface. Thegold electrode bearing 11-hydroxy-1-undecanethiol (11-HUT) as animmobilized insulating molecule showed a smaller slope of the currentversus the applied potential than that of the untreated gold electrode.This means that the electrode surface has an increased insulatingproperty by immobilizing an insulating molecule to the electrodesurface. In addition, the gold electrode bearing 11-HUT as animmobilized insulating molecule showed a smaller absolute value of thecurrent than that of the untreated gold electrode. This is because theuntreated gold electrode has a layer called “electric double layer”having a thickness substantially corresponding to one molecule andthereby shows a large electrostatic capacity of about 14 μF/cm²; but, incontrast, the gold electrode bearing the immobilized insulating moleculehas an insulating layer having a thickness of about 2 nm correspondingto the length of the insulating molecule and thereby shows a decreasedelectrostatic capacity of about 2.3 μF/cm². Thus, by immobilizing aninsulating molecule to a surface of an electrode, the electrode has anincreased surface resistance and thereby shows a decreased electrostaticcapacity. As a result, the leakage current is reduced, a chargingcurrent is also reduced, and the measuring electrode can detect afurther smaller variation in concentrations of redox compounds.

Effects of the immobilization of an electrochemically active material toa surface of a measuring electrode through an insulating molecule willbe illustrated with reference to still another embodiment of the presentinvention. FIG. 7 illustrates an evaluation system for a measuringelectrode bearing an electrochemically active material immobilizedthereto through an insulating molecule. This evaluation system includesa potentiometer 701, a reference electrode 702, a sample solutioninjector 703 that supplies a sample solution containing a targetmaterial, and a measuring cell 704. The measuring cell 704 contains ameasuring solution 707. A gold electrode 708 and the reference electrode702 are arranged in the measuring solution 707. The gold electrode 708bears an electrochemically active material 705 immobilized theretothrough an insulating molecule 706.

FIG. 8 shows how the potential difference between the referenceelectrode and the gold electrode varies depending on the time elapsedfrom the injection of a target material, which was measured using theevaluation system in FIG. 7. The reference electrode 702 used herein wasa Ag/AgCl reference electrode containing a saturated aqueous potassiumchloride solution as an internal fluid. The electrochemically activematerial 705 immobilized through the insulating molecule 706 was (a)6-ferrocenyl-1-hexanethiol, (b) 8-ferrocenyl-octanethiol, and (c)11-FUT, respectively. The sample solution in the sample solutioninjector was an aqueous potassium ferrocyanide solution. The measuringsolution 807 was a 0.1 M aqueous sodium sulfate solution. In FIG. 8, theabscissa indicates the time elapsed from the injection of the samplesolution, and the ordinate indicates the potential difference betweenthe reference electrode and the gold electrode. The potential differencewas normalized by defining the potential immediately before theinjection of the sample solution as 1 and defining the potential 100seconds after the injection as 0. The time for the potential to be 0.1after the injection of the sample solution, namely, the time for thevariation in potential to be 90% was defined as a relaxation time. Therelaxation times in (a), (b), and (c) satisfy the following condition:(a)>(b)>(c). Specifically, the relaxation time decreases with anincreasing length of the insulating molecule 706. The insulatingproperty between the measuring electrode and the measuring solutionincreases with an increasing length of the insulating molecule 706. Inother words, the response speed increases with an increasing insulatingproperty.

The sensitivity can be improved by selecting an electrochemically activematerial to be immobilized according to a redox compound to be used.This will be explained with reference to another embodiment of thepresent invention. FIG. 9 shows data of the measurement of NAD and NADHas redox compounds using an electrode including a gold electrode bearing11-FUT immobilized on its surface (11-FUT-immobilized electrode). FIG.10 shows data of the measurement of NAD and NADH as redox compoundsusing an electrode including a gold electrode bearing11-amino-1-undecanethiol immobilized on its surface and further bearingpyrroloquinoline quinone (PQQ) immobilized thereon (PQQ-immobilizedelectrode). The 11-FUT-immobilized electrode has a slope sensitivity of24 mV, and the PQQ-immobilized electrode has a slope sensitivity of 58mV.

The above-mentioned reactions will be theoretically considered. When the11-FUT-immobilized electrode is used, the following equilibrium reactionestablishes between the immobilized ferrocene and NAD/NADH in thesolution.

2 Ferrocene (reduced)+NAD

2 Ferrocene (oxidized)+NADH

Accordingly, the following equation establishes:

$\begin{matrix}{\left\lbrack {{Equation}\mspace{14mu} 1} \right\rbrack \;} & \; \\{\left( \frac{\left\lbrack {{Ferrocene}({oxidized})} \right\rbrack}{\left\lbrack {{Ferrocene}({reduced})} \right\rbrack} \right)^{2} = \frac{\lbrack{NAD}\rbrack}{\lbrack{NADH}\rbrack}} & (1)\end{matrix}$

[Ferrocene (oxidized)]: Oxidized ferrocene concentration

[Ferrocene (reduced)]: Reduced ferrocene concentration

[NAD]: NAD concentration

[NADH]: NADH concentration

The surface potential E of the electrode is theoretically-determinedfrom the ratio between oxidized form and reduced form of theelectrochemically active material immobilized to the electrode surface,according to the following Nernst's equation:

$\begin{matrix}{\left\lbrack {{Equation}\mspace{14mu} 2} \right\rbrack \;} & \; \\{E = {E^{0} + {\frac{RT}{n\; F}\log \frac{\lbrack{Ox}\rbrack}{\lbrack{Red}\rbrack}}}} & (2)\end{matrix}$

E⁰: Standard oxidation-reduction potentialR: Gas constantT: Absolute temperaturen: Number of electrons to be exchangedF: Faraday constant[Red]: Concentration of reduced electrochemically active material[Ox]: Concentration of oxidized electrochemically active material

Equation (1) is substituted into Equation (2) to give Equation (3):

$\begin{matrix}{\left\lbrack {{Equation}\mspace{14mu} 3} \right\rbrack \;} & \; \\{E = {E^{0} + {\frac{RT}{2\; F}\log \frac{\lbrack{NAD}\rbrack}{\lbrack{NADH}\rbrack}}}} & (3)\end{matrix}$

The slope sensitivity to the NAD and NADH concentrations is thusdetermined as 30 mV at 25° C. The measured slope sensitivity was smallerthan this theoretical value. This is probably because a redox reactionbetween ferrocene and NAD/NADH has a low reaction rate and thereby didnot reach an equilibrium state.

When the PQQ-immobilized electrode is used, the following equilibriumreaction establishes between the immobilized PQQ and NAD/NADH in thesolution.

PQQ (reduced)+NAD

G PQQ+NADH

Accordingly, the following equation establishes:

$\begin{matrix}{\left\lbrack {{Equation}\mspace{14mu} 4} \right\rbrack \;} & \; \\{\frac{\left\lbrack {{PQQ}({oxidized})} \right\rbrack}{\left\lbrack {{PQQ}({reduced})} \right\rbrack} = \frac{\lbrack{NAD}\rbrack}{\lbrack{NADH}\rbrack}} & (4)\end{matrix}$

[PQQ (oxidized)]: Oxidized PQQ concentration[PQQ (reduced)]: Reduced PQQ concentration

Equation (4) is substituted into Equation (2) to give Equation (5):

$\begin{matrix}{\left\lbrack {{Equation}\mspace{14mu} 5} \right\rbrack \;} & \; \\{E = {E^{0} + {\frac{RT}{F}\log \frac{\lbrack{NAD}\rbrack}{\lbrack{NADH}\rbrack}}}} & (5)\end{matrix}$

□□□□□□□□□□□

The slope sensitivity to the NAD and NADH doncentrations is thusdetermined as 59 mV at 25° C. The measured slope sensitivity wassubstantially equal to the theoretical value, indicating that a redoxreaction between PQQ and NAD/NADH reached an equilibrium state when thepotential was measured, because it has a sufficiently high reactionrate. The sensitivity can be improved by selecting an electrochemicallyactive material to be immobilized to a surface of an electrode accordingto a redox compound to be used in this manner. Suitable combinations forelectrically detecting pyrophosphoric acid will be illustrated below, byway of example.

In case that a Ferrocene is the immobilized electrochemically activemolecule, the metal complex is better for the redox compound, especiallya transition metal, specifically whose d-orbitals are not full, forexample Fe, Ru, and Co.

TABLE 1 Immobilized electrochemically Redox active molecule compoundReaction system (example) Ferrocene Ferricyanide, FerrocyanidePyrophosphoric acid + Fructose

Fructose 1,6-dihosphate + Phosphoric acid

Glyceraldehyde-3-phosphate + Dihydroxyacetone phosphateGlyceraldehyde-3-phosphate + NAD + Phosphoric

Glycerate-1,3-diphosphate + NADH NADH + 2

NAD + 2 Ferrocyanide PQQ NAD, NADH Pyrophosphoric acid +

Pi + Pyruvic acid +ATP Pyruvic acid +

Lactic acid + NAD

FIG. 11 shows measured data in detection of an extension reaction ofDNA, and calculated data thereof. To a solution were sequentially addeddTTP, dATP, and dGTP (each 5 mM, 1 μL). The solution contained a mixtureof a double-stranded DNA (10 μL, 50 μL), DNA polymerase (50 U/ml, 1 μL),fructose 6-phosphate kinase (0.5 U/ml, 1 μL), aldolase (50 U/ml, 1 μL),glyceraldehyde-3-phosphate dehydrogenase (1 U/ml, 1 μL), diaphorase (10U/ml, 1 μL), fructose 6-phosphate (90 mM, 1 μL), NAD (3 mg/ml, 1 μL),and potassium ferricyanide (10 mM, 5 μL). A primer DNA and a sample DNAused as the double-stranded DNA had the following sequences:

Primer DNA:  3′-CACAC TCACA GTTTT CACTT-5′ Sample DNA: 3′-GCATA CACTA AAGTG AAAAC TGTGA GTGTG-5′

When dTTP was added, a single-base extension reaction occurred by theaction of a DNA polymerase. This is because the base at position 10 fromthe 3′ end of the sample DNA is A (adenine). Pyrophosphoric acid formedas a result of the extension reaction caused reduction of ferricyanideto yield ferrocyanide according to the following reactions:

Pyrophosphoric acid+Fructose 6-phosphate→Fructose1,6-diphosphate+Phosphoric acid

Fructose 1,6-diphosphate→Glyceraldehyde-3-phosphate+Dihydroxyacetone

Glyceraldehyde-3-phosphate+NAD+Phosphoricacid→Glycerate-1,3-diphosphate+NADH

NADH+2 Ferricyanide→NAD+2 Ferrocyanide

The formation of ferrocyanide caused a variation in surface potential ofthe electrode. As a result, a decrease in potential of 16.7 mV wasobserved (FIG. 11). Next, dATP was added, and a single-base extensionreaction occurred. This is because the base at position 9 from the 3′end of the sample DNA is T (thymine). This extension reaction caused adecrease in potential of 15.1 mV as observed in the same manner asabove. Next, dGTP was added. As a result, the solution contained dATP,dTTP, and dGTP as dNTPs, and there occurred extension reactions ofGTGTATG, which is a complementary sequence of the base sequence CATACACat positions 2 to 8 from the 3′ end of the sample DNA, except for thebase G at the 3′ end. The extension reactions of the seven bases causeda decrease in potential of 23.8 mV.

The decrease in potential will be theoretically considered. When thesolution has a ferrocyanide concentration “x”, a potential of E₀−59 logx occurs on the surface of the electrode at 25° C. according to theNernst's equation. When ferrocyanide is formed in a concentration of Δxas a result of a single-base extension reaction, and ferrocyanide has aconcentration of c₀ before the extension reaction, ferrocyanideconcentrations at respective points of time are as follows:

Before extension reaction:

After addition of dTTP: c₀+Δx

After addition of dATP: c₀+2Δx

After addition of dGTP: c₀+9Δx

Accordingly, potentials on surface of the electrode at the respectivepoints of time are as follows:

Before extension reaction: E₀−59 log(c₀)

After addition of dTTP: E₀−59 log(c₀+Δx)

After addition of dATP: E₀−59 log(c₀+2Δx)

After addition of dGTP: E₀−59 log(c₀+9Δx)

Consequently, variations in potential are:

Addition of dTTP: 59 log(c₀+Δx)−59 log(c₀)

Addition of dATP: 59 log(c₀+2Δx)−59 log(c₀+Δx)

Addition of dGTP: 59 log(c₀+9Δx)−59 log(c₀+2Δx)

When fitting was conducted, an optimum solution was obtained at Δx of0.83c (FIG. 11). The calculated data satisfactorily meet with themeasured data, showing that pyrophosphoric acid formed as a result ofextension reactions of the double-stranded DNA by adding respectivedNTPs was detected as variations in potential.

SNPs (single nucleotide polymorphisms) can be detected in the samemanner. Specifically, dCTP and dTTP (each 5 mM, 1 μL) were sequentiallyadded to a solution containing a double-stranded DNA (50 μM, 50 μL), DNApolymerase (50 U/ml, 1 μL), fructose 6-phosphate kinase (0.5 U/ml, 1μL), aldolase (50 U/ml, 1 μL), glyceraldehyde-3-phosphate dehydrogenase(1 U/ml, 1 μL), diaphorase (10 U/ml, 1 μL), fructose 6-phosphate (90 mM,1 μL), NAD (3 mg/ml, 1 μL), and potassium ferricyanide (10 mM, 5 μL). Aprimer DNA and sample DNAs used as the double-stranded DNA had thefollowing sequences:

Primer DNA:  3′-CACAC TCACA GTTTT CACTT-5′ Sample DNA (wild-type): 3′-GCATA CACTA AAGTG AAAAC TGTGA GTGTG-5′ Sample DNA (mutant): 3′-GCATA CACTG AAGTG AAAAC TGTGA GTGTG-5′

Measurements were conducted on a combination of the primer DNA and thesample DNA (wild-type) and on a combination of the primer DNA and thesample DNA (mutant), respectively. With reference to FIG. 12, when theprimer DNA and the sample DNA (wild-type) were used in combination,decreases in potential of 1 mV or less and 19.5 mV were observed byadding dCTP and dTTP, respectively. In contrast, when the primer DNA andthe sample DNA (mutant) were used in combination, decreases in potentialof 20.2 mV and 1 mV or less were observed by addition of dCTP and dTTP,respectively. These results reflect that, when the primer DNA and thesample DNA (wild-type) are used in combination, an extension reactionoccurs not by the addition of dCTP but by the addition of dTTP, and thatwhen the primer DNA and the sample DNA (mutant) are used in combination,an extension reaction occurs not by the addition of dTTP but by theaddition of dCTP. By using these, SNPs can be detected.

When smaller volumes of samples are to be measured according to knowntechnologies, signals may be lowered. However, this can be prevented bymeasuring samples according to potentiometry. This will be theoreticallyconsidered as follows. How data according to luminometry (detection ofphotoluminescence) and potentiometry vary depending on the bead radiuswill be considered, assuming that a bead having a specific surface area(ratio of actual surface area to geometrical surface area) R and aradius r is arranged in a cubic cell having a side of 2r. The surfacearea of the bead is 4πr²R and is proportional to the square of theradius. According to luminometry, the signal intensity varies dependingon the amount of DNA, namely, the surface area of the bead, and isproportional to the square of the bead radius when DNA is immobilized ata constant density. Accordingly, the signal intensity decreases with adecreasing volume of a sample to be measured according to luminometry.In contrast, the signal intensity according to potentiometry variesdepending on the concentration of DNA. The volume of a sample solutionis (8−4/3π)r³ which is obtained by subtracting the volume of the bead4/3πr³ from the volume of the cubic cell 8r³ and is proportional to thecube of the bead radius. Accordingly, the signal intensity according topotentiometry is proportional to the reverse number of the bead radius,because the concentration of DNA is inversely proportional to the beadradius when DNA is immobilized at a constant density. Consequently, thesignal intensity increases with a decreasing volume of the sample to bemeasured according to potentiometry. Thus, the potentiometry is suitablefor massive parallel analyses, because it is free from decrease insignal intensity even if smaller amounts of samples are measured.

Which bead radius is suitable will be considered below. Theabove-mentioned calculation shows that the volume of the sample solutionis (8−4/3π)r³. When DNA molecules are immobilized to the bead surface atintervals d, 4πr²R/d² DNA molecules are immobilized to the bead surface.The DNA concentration in this case is 3R/{(6−π)d²rN_(A)}mol/m³, and therelationship between the bead radius and the DNA concentration isdetermined as shown in FIG. 13 assuming that R is 1. When a minimumdetectable concentration of a redox compound is 10⁻⁶M, an extensionreaction of one base can be detected at a bead radius of 70 μm or less,namely, at a bead diameter of 140 μm or less.

While preferred embodiments have been described, it should be understoodby those skilled in the art that various modifications, combination,sub-combinations, and alternations may occur depending on designrequirements and other factors insofar as they are within the scope ofthe appended claims or the equivalents thereof.

1. A deoxyribonucleic acid (DNA) analysis method comprising the stepsof: providing a sample containing a DNA to be analyzed and a primerhybridized to the DNA; carrying out an extension reaction of the primerin the presence of deoxyribonucleotide triphosphates (dNTPs) and a DNApolymerase so that a primer extension reaction product is formed;converting pyrophosphoric acid formed as a result of the extensionreaction into a redox compound; and electrically detecting the redoxcompound.
 2. The DNA analysis method of claim 1, wherein the step ofdetecting comprises detecting a variation in interface potential of anelectrode in contact with the sample.
 3. The DNA analysis method ofclaim 2, wherein a variation in interface potential of the electrode isdetected using a field-effect transistor, wherein the field-effecttransistor comprises a gate terminal electrically connected to theelectrode, and wherein the field-effect transistor and the electrode arearranged on or above the same substrate.
 4. The DNA analysis method ofclaim 2, wherein the dNTPs are deoxyadenosine triphosphate (dATP),deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP),and deoxythymidine triphosphate (dTTP), or sulfur-containing derivativesof the dNTPs.
 5. The DNA analysis method of claim 4, wherein one of thedNTPs is a sulfur-containing deoxyribonucleotide triphosphate (dNTP). 6.The DNA analysis method of claim 2, wherein an electrochemically activematerial is immobilized to the electrode through an insulating molecule.7. The DNA analysis method of claim 6, wherein the insulating moleculecomprises a carbon chain.
 8. The DNA analysis method of claim 6, whereinthe redox compound is oxidized nicotinamide adenine dinucleotide (NAD),and wherein the electrochemically active material is aquinone-containing molecule.
 9. The DNA analysis method of claim 6,wherein the redox compound is a metal complex, and wherein theelectrochemically active material is a ferrocene-containing material.10. The DNA analysis method of claim 6, further comprising the step ofcarrying out washing a vessel for measuring with a washing buffer beforecarrying out said extension reaction.
 11. The DNA analysis method ofclaim 10, wherein the washing buffer comprises a redox compound forinitialization.
 12. The DNA analysis method of claim 10, wherein thewashing buffer comprises thiol compound, hydrogen peroxide, or potassiumferrocyanide.